Key Determinants for Calcium Binding Affinity of EF-Hand Proteins

0092486 Yang本项目的具体目标是了解EF-手环的位点特异性钙结合特性，并确定影响蛋白质EF-手基序钙亲和力的关键因素。钙结合是EF-手蛋白在控制细胞钙水平和通过钙诱导的构象变化调节许多细胞过程中的生物学功能所必需的。关于个别EF-手基序的钙结合特性的位点特异性信息对于定义钙结合过程的序列和每个EF-手基序对完整蛋白质的构象变化的贡献是必不可少的。在这项研究中，PI将通过将单个EF-钙结合位点工程化到支架蛋白中来研究分离的EF-手基序的内在钙结合亲和力。 这种方法特别侧重于一个单独的EF-手基序的内在结合能力，而没有遇到的复杂性，在合作，多网站系统的天然EF-手蛋白。 为了获得EF-手环的钙结合特性的位点特异性信息，EF-手基序的构建块将被系统地引入宿主支架中。为了确定带电配体对EF-环的钙亲和力的贡献，分离的EF-环中带负电荷的配体的数量和几何排列也将变化。

0092486YangThe specific goals of this project are to understand site-specific calcium binding properties of EF-hand loops and identify the key factors contributing to calcium affinity of EF-hand motifs in proteins. Calcium binding is essential for the biological functions of EF-hand proteins in controlling cellular calcium levels and in regulating many cellular processes through calcium-induced conformational change. Site-specific information about calcium binding properties of individual EF-hand motifs is essential for defining the sequence for the calcium binding process and the contribution of each EF-hand motif to the conformational change of the intact protein. In this research, the PI will investigate the intrinsic calcium binding affinity of isolated EF-hand motifs by engineering a single EF-calcium-binding site into a scaffold protein. This approach focuses specifically on the intrinsic binding ability of an individual EF-hand motif without the complexities encountered in cooperative, multi-site systems of natural EF-hand proteins. To obtain site-specific information about calcium-binding properties of EF-hand loops, the building blocks of EF-hand motifs will be systematically introduced into the host scaffold. To identify contributions of charged ligands to calcium affinity of EF-loops, the number and geometric arrangement of negatively charged ligands in isolated EF-loops will be also varied.